Imaging Beta-Amyloid Peptides and Inhibition of Beta-Amyloid Peptide Aggregation

ABSTRACT

The present invention is in the field of pharmaceuticals and chemical industries. In particular, the present invention relates to methods for labeling, imaging and detecting the beta-amyloid (Aβ) peptides, oligomers, and fibrils in vitro by using carbazole-based fluorophores. A further aspect of the present invention relates to a method of reducing and preventing aggregation of beta-amyloid peptides for Alzheimer&#39;s disease (AD) as well as of treating and/or preventing Alzheimer&#39;s disease by using carbazole-based fluorophores.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part application of the USnon-provisional patent application Ser. No. 13/447,127 filed Apr. 13,2012, which claims priority of U.S. provisional application No.61/477,614 filed Apr. 21, 2011, and which the disclosure is herebyincorporated by reference in its entirety.

FIELD OF INVENTION

The present invention relates to methods of detecting and monitoringaggregation of beta-amyloid peptides which are associated withneurodegenerative diseases as well as treating and/or preventing theneurodegenerative diseases by using carbazole-based fluorophores. Inparticular, the present invention provides methods for labeling andimaging the beta-amyloid (Aβ) peptides, oligomers, and fibrils in vitroand/or in vivo, as well as treating and/or preventing Alzheimer'sdisease by using the carbazole-based fluorophores of the presentinvention.

BACKGROUND OF INVENTION

Loss of memory and cognitive functions are often associated with aging.This is the result of neurodegeneration. However, in some cases, thisprocess of neurodegeneration becomes accelerated due to premature celldeath in the brain, leading to a variety of cognitive impairments ordementia. Among these neurodegenerative disorders, Alzheimer's disease(AD) is most prevalent in recent years. It has also attractedconsiderable attention locally because Prof. Charles K. Kao, formerpresident of the Chinese University of Hong Kong and Nobel Laureate inPhysics, 2009, was stricken with this devastating disease.

More than 36 million people worldwide were estimated to suffer fromAlzheimer's disease (AD) in 2009 and the patient number was expected toincrease to 115 million in 2050. The incidence rate of AD is known toincrease with age. At age over 65, the incidence rate is about 5% in thegeneral population. At age over 80, the incidence rate increases toabout 20%, i.e., one in five. Current drug treatments can only improvesymptoms and produce no profound cure. In recent years, severalapproaches aimed at inhibiting disease progression have advanced toclinical trials. Among them, strategies targeting the production andclearance of the Aβ peptide, which is thought to be a critical proteininvolved in the pathogenesis of the disease, are the most advanced.

Aβ peptide is the principal protein component of the Aβ plaques, whichare found in the brains of AD patients during autopsy. The occurrence ofthe Aβ plaques, considered a cardinal feature of AD, provides the onlyconfirmed diagnosis of the disease. Extensive researches in past decadeshave indicated a central role for the Aβ peptide in the disease processwhere the Aβ peptides assemble (aggregate) into Aβ fibrils which exert acytotoxic effect towards the neurons and initiate the pathogeniccascade. Recent studies showed that oligomeric, prefibrillar anddiffusible assemblies of Aβ peptides are also deleterious. The abilityof this peptide to form Aβ fibrils seems to be largelysequence-independent, and many proteins can form structures with thecharacteristic cross-β stacking perpendicular to the long axis of thefiber. Although a consensus mechanism for the pathogenic oligomericassembly has yet to emerge, the idea of finding some brain-penetratingsmall molecules that can interfere with the interactions among the Aβpeptide monomers and thus inhibit the formation of the neurotoxicoligomers and the resulting Aβ plaques is an attractive approach totreating/preventing the disease. The use of agents that stabilize themonomer, interfere with the aggregation process (amyloidogenesis) andallow for the isolation of the intermediate species will help toelucidate the molecular mechanism of Aβ fibril formation. In addition,imaging agents that can specifically bind Aβ fibrils and plaques invitro and in vivo are of paramount importance for studying thepathological events of the disease, disease diagnosis and monitoring oftherapeutic treatment.

We have previously shown that carbazole-based fluorophores are highlysensitive fluorescent light-up probe for double strand DNA and stronglyactive two-photon absorption dyes for two-photon excited bioimaging, thedisclosure of which is incorporated by reference herein. Recently, themono-cyanine fluorophore has also been found to exhibit binding affinitytowards beta amyloid (Aβ) peptide concomitant with strong fluorescentenhancement. These findings provide us the lead molecular structure todesign and synthesize novel functional carbazole-based fluorophores forimaging and inhibition the aggregation of Aβ peptides.

Citation or identification of any reference in this section or any othersection of this application shall not be construed as an admission thatsuch reference is available as prior art for the present application.

SUMMARY OF INVENTION

Accordingly, the first objective of the presently claimed inventionrelates to methods for labeling, imaging and detecting the beta-amyloid(Aβ) peptides, oligomers, and fibrils in vitro by using carbazole-basedfluorophores.

In a first aspect of the present invention there is provided a method oflabeling, imaging and detecting beta-amyloid (Aβ) peptides, oligomersand fibrils using carbazole-based fluorophores comprising a formula of Sor V series:

wherein Ar is a heteraromatic ring selected from the group consisting ofpyridinyl, substituted pyridinyl, quinolinyl, substituted quinolinyl,acridinyl, substituted acridinyl, benzothiazolyl, substitutedbenzothiazolyl, benzoxazolyl, and substituted benzoxazolyl;R₁ is selected from the group consisting of polyethylene glycol chain,alkyl, substituted alkyl, peptide chain, glycosidyl, andC(O)NHCH((CH₂CH₂O)₂CH₃)₂;R₂ is selected from the group consisting of ethenyl, ethynyl, azo andazomethinyl.R₃ is selected from the group consisting of alkyl, HO-alkyl, HS-alkyl,H₂N-alkyl, HN-alkyl-alkyl, HOOC-alkyl, (alkyl)₃N⁺-alkyl, and(Ph)₃P⁺-alkyl; andX is an anion selected from the group consisting of F⁻, Cl⁻, Br⁻, I⁻,HSO₄ ⁻, H₂PO₄ ⁻, HCO₃ ⁻, tosylate, and mesylate.

In a first embodiment of the first aspect of the present invention thereis provided a method wherein said Ar is selected from a quinolinyl orsubstituted quinolinyl; said R₁ is a 2-(2-methoxyethoxy)ethoxy; said R₂is an ethenyl; said R₃ is selected from a methyl, 2-hydroxyethyl, ethylor 3-hydroxypropyl; and said X is selected from a chloride, bromide oriodide, the carbazole-based fluorophores thereof are represented by theformula SLM, SLOH, SLE and SLOH-Pr:

In a second embodiment of the first aspect of the present inventionthere is provided a method wherein said Ar is selected from a quinolinylor substituted quinolinyl; said R1 is a methyl; said R2 is an ethenyl;said R3 is a methyl; and said X is selected from a chloride, bromide oriodide, and the carbazole-based fluorophores thereof are represented bythe formula Me-SLM:

In a third embodiment of the first aspect of the present invention thereis provided a method wherein said Ar is selected from an acridinyl orsubstituted acridinyl; said R₁ is a 2-(2-methoxy-ethoxy)ethoxy; said R₂is an ethenyl; said R₃ is selected from a methyl or 2-hydroxyethyl; andsaid X is selected from a chloride, bromide or iodide, and thecarbazole-based fluorophores thereof are represented by the formula SAMand SAOH:

In a fourth embodiment of the first aspect of the present inventionthere is provided a method wherein said Ar is selected from a pyridinylor substituted pyridinyl; said R₁ is a 2-(2-methoxyethoxy)ethoxy; saidR₂ is an ethenyl; said R is selected from a methyl or 2-hydroxyethyl;and said X is selected from a chloride, bromide or iodide, thecarbazole-based fluorophores thereof are represented by the formula SPMand SPOH:

In a fifth embodiment of the first aspect of the present invention thereis provided a method wherein said carbazole-based fluorophores areconjugated with paramagnetic metal complexes comprising gadolinium(III),iron(III), manganese(II) complexes, Gd(III)-based chelates, and anycomplexes which are detected by magnetic resonance imaging.

In a sixth embodiment of the first aspect of the present invention thereis provided a method, wherein the carbazole-based fluorophores arewater-soluble and/or are non-toxic.

In a seventh embodiment of the first aspect of the present inventionthere is provided a method wherein the carbazole-based fluorophores areable to pass through the blood-brain barrier.

In an eighth embodiment of the first aspect of the present inventionthere is provided a method wherein the carbazole-based fluorophores areadministered in vitro and/or in vivo.

In the second aspect of the present invention there is provided a methodof labelling, imaging and detecting beta-amyloid (Aβ) peptides,oligomers, and fibrils using carbazole-based fluorophores comprising aformula S series:

-   wherein Ar is a heteraromatic ring selected from the group    consisting of pyridinyl, substituted pyridinyl, quinolinyl,    substituted quinolinyl, acridinyl, substituted acridinyl,    benzothiazolyl, substituted benzothiazolyl, benzoxazolyl, and    substituted benzoxazolyl;-   R₁ is a radical selected from the group consisting of polyethylene    glycol chain, alkyl, substituted alkyl, peptide chain, glycosidyl,    and C(O)NHCH((CH₂CH₂O)₂CH₃)₂;-   R₂ is selected from the group consisting of ethenyl, ethynyl, azo    and azomethinyl.-   R₃ is a radical selected from the group consisting of HO-alkyl,    alkyl-COOalkyl, alkyl-CONH₂, alkyl-CONHalkyl, polyethylene glycol    chain;-   X is an anion selected from the group consisting of F, Cl, Br, I,    HSO₄, H₂PO₄, HCO₃, tosylate, and mesylate;-   Y is selected from the group consisting of H, F, Cl, OH, and OCH₃.

In a first embodiment of the second aspect of the present inventionthere is provided a method wherein Ar is selected from a quinolinyl orsubstituted quinolinyl; said R₁ is a 2-(2-methoxyethoxy)ethoxy; said R₂is an ethenyl; R₃ is a 2-hydroxyethyl or acetamide or acetate or2-(2-methoxyethoxy)ethoxy; said X is a chloride or bromide or iodide andsaid Y is a H or F which are represented by the formula F-SLOH, SLAD,SLAce, and SLG:

In a second embodiment of the second aspect of the present inventionthere is provided a method wherein said carbazole-based fluorophores areconjugated with paramagnetic metal complexes comprising gadolinium(III),iron(III), manganese(II) complexes, Gd(III)-based chelates, and anycomplexes which are detected by magnetic resonance imaging.

In a third embodiment of the second aspect of the present inventionthere is provided a method wherein the carbazole-based fluorophores arenon-toxic and/or are able to pass through the blood-brain barrier.

In a fourth embodiment of the second aspect of the present inventionthere is provided a method wherein the carbazole-based fluorophores areadministered in vitro and/or in vivo.

A further embodiment of the first and/or second aspect of the presentinvention wherein said carbazole-based fluorophores are used in imagingtechniques comprising magnetic resonance imaging, positron emissiontomography, near-infrared fluorescence imaging and multiphoton excitedimaging.

Throughout this specification, unless the context requires otherwise,the word “include” or “comprise” or variations such as “includes” or“comprises” or “comprising”, will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers. It is also noted that in thisdisclosure and particularly in the claims and/or paragraphs, terms suchas “included”, “comprises”, “comprised”, “comprising” and the like canhave the meaning attributed to it in U.S. patent law; e.g., they canmean “includes”, “included”, “including”, and the like; and that termssuch as “consisting essentially of” and “consists essentially of” havethe meaning ascribed to them in U.S. patent law, e.g., they allow forelements not explicitly recited, but exclude elements that are found inthe prior art or that affect a basic or novel characteristic of thepresent invention.

Furthermore, throughout the specification and claims, unless the contextrequires otherwise, the word “include” or variations such as “includes”or “including”, will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers.

Other definitions for selected terms used herein may be found within thedetailed description of the present invention and apply throughout.Unless otherwise defined, all other technical terms used herein have thesame meaning as commonly understood to one of ordinary skill in the artto which the present invention belongs.

Other aspects and advantages of the present invention will be apparentto those skilled in the art from a review of the ensuing description.

BRIEF DESCRIPTION OF DRAWINGS

The above and other objects and features of the present invention willbecome apparent from the following description of the present invention,when taken in conjunction with the accompanying drawings, in which:

FIG. 1 shows the fluorescence spectra of SPM, SPOH, SLM, SLOH, SLE,SLOH-Pr, Me-SLM, SAM, and SAOH (1 μM) in phosphate buffer upon additionof various concentrations of Aβ (1-40) fibrils prepared from Aβ₄₀ withseed incubated at 37° C. for an hour in buffer (left column). FIG. 1Ashows the fluorescence spectra of SPM in various concentrations of Aβ(1-40) fibrils. FIG. 1C shows the fluorescence spectra of SPOH invarious concentrations of Aβ (1-40) fibrils. FIG. 1E shows thefluorescence spectra of SLM in various concentrations of Aβ (1-40)fibrils. FIG. 1G shows the fluorescence spectra of SLOH in variousconcentrations of Aβ (1-40) fibrils. FIG. 1I shows the fluorescencespectra of SLE in various concentrations of Aβ (1-40) fibrils. FIG. 1Kshows the fluorescence spectra of SLOH-Pr in various concentrations ofAβ (1-40) fibrils. FIG. 1M shows the fluorescence spectra of Me-SLM invarious concentrations of Aβ (1-40) fibrils.

FIG. 1O shows the fluorescence spectra of SAM in various concentrationsof Aβ(1-40) fibrils. FIG. 1Q shows the fluorescence spectra of SAOH invarious concentrations of Aβ (1-40) fibrils. FIG. 1 also shows thefluorescence spectra of SPM, SPOH, SLM, SLOH, SLE, SLOH-Pr, Me-SLM, SAM,and SAOH (1 μM) in phosphate buffer itself, in the presence of 100 equvof Aβ₄₀ in monomeric form, and in the presence of 100 equv of Aβ₄₀ infibril state in phosphate buffer (right column). FIG. 1B shows thefluorescence spectra of SPM on its own and in the presence of twodifferent forms of Aβ₄₀. FIG. 1D shows the fluorescence spectra of SPOHon its own and in the presence of two different forms of Aβ₄₀. FIG. 1Fshows the fluorescence spectra of SLM on its own and in the presence oftwo different forms of Aβ₄₀. FIG. 1H shows the fluorescence spectra ofSLOH on its own and in the presence of two different forms of Aβ₄₀. FIG.1J shows the fluorescence spectra of SLE on its own and in the presenceof two different forms of Aβ₄₀. FIG. 1L shows the fluorescence spectraof SLOH-Pr on its own and in the presence of two different forms ofAβ₄₀. FIG. 1N shows the fluorescence spectra of Me-SLM on its own and inthe presence of two different forms of Aβ₄₀. FIG. 1P shows thefluorescence spectra of SAM on its own and in the presence of twodifferent forms of Aβ₄₀. FIG. 1R shows the fluorescence spectra of SAOHon its own and in the presence of two different forms of Aβ₄₀.

FIG. 2 shows the fluorescence spectra of SPM, SPOH, SLM and SLOH inphosphate buffer (1 μM) upon addition of various concentrations of Aβ(1-40) and Aβ(1-42), respectively. FIG. 2A shows the fluorescencespectra of SPM in various concentrations of Aβ(1-40). FIG. 2B shows thefluorescence spectra of SPM in various concentrations of Aβ(1-42). FIG.2C shows the fluorescence spectra of SPOH in various concentrations ofAβ(1-40). FIG. 2D shows the fluorescence spectra of SPOH in variousconcentrations of Aβ(1-42). FIG. 2E shows the fluorescence spectra ofSLM in various concentrations of Aβ(1-40). FIG. 2F shows thefluorescence spectra of SLM in various concentrations of Aβ(1-42). FIG.2G shows the fluorescence spectra of SLOH in various concentrations ofAβ(1-40).

FIG. 2H shows the fluorescence spectra of SLOH in various concentrationsof Aβ(1-42).

FIG. 3 shows TIRFM images of Aβ fibrils after incubation with thecarbazole-based fluorophores, SPM (FIG. 3A) excited at 445 nm and SLM(FIG. 3B) and SLOH (FIG. 3C) excited at 488 nm, respectively.

FIG. 4 shows in vitro fluorescence imaging of neuronal cells by usingthe carbazole-based fluorophore, SLOH (FIG. 4A) The lambda scans of theimages match well with the fluorescence spectrum of the SLOH (FIG. 4B).

FIG. 5 shows absorption and fluorescence spectra of the carbazole-basedfluorophores, SAM (FIG. 5A) and SAOH (FIG. 5B) in phosphate buffersolution.

FIG. 6 shows CD spectra of Aβ(1-40) peptide (FIG. 6A) and fibrils (FIG.6B) in the absence and presence of SLOH (1:1) (20 μM).

FIG. 7 shows TIRFM images of Aβ fibrils (FIG. 7A) and Aβ peptide afterincubation with the carbazole-based fluorophore, SLOH (FIG. 7B), SAOH(FIG. 7C), SLE (FIG. 7D), SLOH-Pr (FIG. 7E), and Me-SLM (FIG. 7F). FIGS.7B, 7C, 7E and 7F show an inhibition of Aβ fibril formation from the Aβmonomer by SLOH and SAOH. These images were obtained by an addition ofThT dye excited at 445 nm.

FIG. 8 shows TEM images of Aβ fibril growth from Aβ peptide (50 μM)seeded for 1 hr at 37° C. in the absence (FIG. 8A) and the presence ofSLOH (FIG. 8B).

FIG. 9 shows ThT, SLM and SLOH fluorescence binding assays for 50 μMAβ₄₀ fibrillation (FIG. 9A). Average length of 1 h incubated Aβ40-fibrilmeasured from TIRFM images after 1 h seed-mediated incubation of Aβ40monomer with (bottom axis, bars) and without (top axis, scatter point)SLOH (50 μM) added at different time points (0, 10, 20, 40 and 60 min)within an one hour-incubation. (FIG. 9B).

FIG. 10 shows cytotoxicities of the carbazole-based SLOH (FIG. 10A),SLOH-Pr (FIG. 10B), Me-SLM (FIG. 10C) and SAOH (FIG. 10D) towards theSH-SY5Y neuronal cell with MTT assays.

FIG. 11 shows cytotoxicities of Aβ peptide monomer (DM/M), oligomers (DMS/MS) and fibrils (DF/F) towards the SH-SY5Y neuronal cell in theabsence and the presence of SLOH (50 μM) (FIG. 11A), SLE (FIG. 11B),SLOH-Pr (FIG. 11C), Me-SLM (FIG. 11D) after 2 hr, 6 hr, and 24 hrincubations.

FIG. 12 shows fluorescence images of transgenic mice brain with tailvein injection of SLOH and co-stained with the Aβ labeling dye, ThT orAβ antibody with DAB stain. Fluorescence image corresponding to SLOHfluorescence captured at 550-630 nm under excitation at 488 nm (FIG.12A); ThT fluorescence captured at 470-550 nm under excitation at 458 nm(FIG. 12B); and overlapped images of previous two images (FIG. 12C). Theoverlapped image revealed the colocalization of fluorescence signals ofSLOH and ThT in cellular components. Differential Interference Contrast(DIC) image of DAB stained brain slide of transgenic mice (FIG. 12D).Fluorescence image of same slide corresponding to SLOH fluorescencecaptured at 550-630 nm under excitation at 488 nm (FIG. 12E); andoverlapped images of previous two images (FIG. 12F). The overlappedimage revealed the colocalization of fluorescence signals of SLOH and Aβantibody in cellular components.

FIG. 13 shows synthesis of carbazole-based fluorophores, SPM, SPOH, SLM,SLOH, SLE, SLOH-Pr, Me-SLM, SAM, and SAOH.

FIG. 14 shows the general chemical structures of carbazole-basedfluorophores, including S series.

FIG. 15 shows the formula “F-SLOH”, “SLAD”, “SLAce”, and “SLG”,respectively.

FIG. 16 shows the fluorescence spectra of F-SLOH (FIG. 16A), SLAD

(FIG. 16B), SLAce (FIG. 16C), and SLG (FIG. 16D) in phosphate buffer (1μM) upon addition of various concentrations of Aβ(1-42) monomer,Aβ(1-40) monomer and Aβ(1-40) fibril, respectively.

FIG. 17 shows the TIRFM images of Aβ monomer and seeds after incubationwith the carbazole-based fluorophores, SLG, F-SLOH, SLAD and SLAce. Thepanels below show the images of seed only and Aβ fibril formationwithout the carbazole-based fluorophore for comparison. These imageswere obtained by an addition of ThT dye excited at 445 nm.

FIG. 18 shows the cytotoxicities of the carbazole-based cyanines,F-SLOH, SLAD, SLAce and SLG towards the SH-SY5Y neuronal cell with MTTassays at 2-hour (FIG. 18A), 6-hour (FIG. 18B) and 24-hourincubation(FIG. 18C).

FIG. 19 shows (FIG. 19A) the influence of F-SLOH, and SLAD suppressionon the toxicity level against various species of Aβ-induced cytotoxicitytowards primary cortical neural cells; (FIG. 19B) shows the reduction ofthe ROS induced by the Aβ species in primary cortical neural cells.

FIG. 20 shows the fluorescence images of mice brain with tail veininjection of SLAD (upper panel) and co-stained with the Aβ labeling dye,and Aβ antibody with DAB stain. Fluorescence image corresponding to SLADfluorescence captured at 550-630 nm under excitation at 488 nm (FIG.20A); Differential Interference Contrast (DIC) image (FIG. 20B); andoverlapped images of previous two images (FIG. 20C). DifferentialInterference Contrast (DIC) image of DAB stained brain slide oftransgenic mice (FIG. 20D). Fluorescence image of same slidecorresponding to SLAD fluorescence captured at 550-630 nm underexcitation at 488 nm (FIG. 20E); and overlapped images of previous twoimages (FIG. 20F). The overlapped image revealed the colocalization offluorescence signals of SLAD and Aβ antibody in cellular components.

FIG. 21 shows the synthesis of carbazole-based fluorophores, F-SLOH,SLAD, SLAce, and SLG. (Note: Reagents and Conditions: a, MeCN, reflux;b, ClCH₂CH₂OCH₂CH₂OCH₃, NaH, DMF, 75° C.; c, NBS, chloroform, 0° C. tor.t.; d, n-BuLi, NFSi, THF, −78° C. to r.t.; e, NBS, chloroform, 0° C.to r.t.; f, n-BuLi, DMF, THF, −78° C. to r.t.; g, MeOH, reflux; h,TMSCl, DMF, 100° C., sealed tube; i, CH₃CH₂OCOCH₂Br, ethanol, r.t.; j,NH₂COCH₂Br, MeCN, reflux.)

DETAILED DESCRIPTION OF THE INVENTION

The presently claimed invention is further illustrated by the followingexperiments or embodiments which should be understood that the subjectmatters disclosed in the experiments or embodiments may only be used forillustrative purpose but are not intended to limit the scope of thepresently claimed invention:

The general chemical structures of carbazole-based fluorophores,including S or V series, are shown as follows:

wherein Ar is a heteraromatic ring selected from the group consisting ofpyridinyl, substituted pyridinyl, quinolinyl, substituted quinolinyl,acridinyl, substituted acridinyl, benzothiazolyl, substitutedbenzothiazolyl, benzoxazolyl, and substituted benzoxazolyl; R₁ is aradical selected from the group consisting of polyethylene glycol chain,alkyl, substituted alkyl, peptide chain, glycosidyl, andC(O)NHCH((CH₂CH₂O)₂CH₃)₂; R₂ is selected from the group consisting ofethenyl, ethynyl, azo and azomethinyl; R₃ is a radical selected from thegroup consisting of alkyl, HO-alkyl, HS-alkyl, H₂N-alkyl, HNalkyl-alkyl,HOOC-alkyl, (alkyl)₃N⁺-alkyl, and (Ph)₃P⁺-alkyl; X is an anion selectedfrom the group consisting of F, Cl, Br, I, HSO₄, H₂PO₄, HCO₃, tosylate,and mesylate.

In one embodiment, Ar is a quinolinyl or substituted quinolinyl; R₁ is a2-(2-methoxyethoxy)ethoxy; R₂ is an ethenyl; R₃ is a methyl,2-hydroxyethyl, ethyl or 3-hydroxypropyl; and X is a chloride, bromideor iodide, and the compounds of which are represented by the aboveformula “SLM”, “SLOH”, “SLE” and “SLOH-Pr”, respectively.

In another embodiment, Ar is a quinolinyl or substituted quinolinyl; R₁is a methyl; R₂ is an ethenyl; R₃ is a methyl; and X is a chloride,bromide or iodide, the compounds of which are represented by the aboveformula Me-SLM.

In a further embodiment, Ar is an acridinyl or substituted acridinyl; R₁is a 2-(2-methoxy-ethoxy)ethoxy; R₂ is an ethenyl; R₃ is a methyl or2-hydroxyethyl; and X is selected from a chloride, bromide or iodide,and the fluorophores of which are represented by the above formula SAMand SAOH, respectively, where the difference between the compounds ofSAM and SAOH is the substitutent at R₃.

In other embodiment, Ar is selected from a pyridinyl or substitutedpyridinyl, R₁ is a 2-(2-methoxyethoxy)ethoxy; R₂ is an ethenyl; R₃ isselected from a methyl or 2-hydroxyethyl; and X is selected from achloride, bromide or iodide, the compounds of which are represented bythe formula SPM and SPOH, respectively.

A novel series of water-soluble carbazole-based fluorophores has beendesigned and developed. These molecules were found to bind to Aβ(1-40)and Aβ(1-42) peptides and, more specifically, their oligomers, andfibrils with strong fluorescence enhancement, therefore allowing directimaging and detection for the Aβ peptides, oligomers and their fibrils(FIG. 1). Upon binding with Aβ peptides, there is about 8- to about82-fold increase in fluorescence intensity concomitant with thesubstantial blue shifts (Δλ=14-22 nm) in the emission spectra of thefluorophores (FIG. 2). Interestingly, the fluorescence enhancement ismuch stronger for fibrils than peptides. (e.g. F_(fibril)/F_(SLOH)=81.5vs. F_(peptide)/F_(SLOH)=6.3). Because of such strong increase influorescence, the signal-to-noise ratio is so high that imaging ofsingle fibrils is possible. (FIG. 3) Compared to common commerciallabeling dyes for Aβ such as Thioflavin-T and Congo Red, thecarbazole-based fluorophores of the present invention provide anadvantage of a wide range of excitation and emission tuning in visibleto infra-red region (FIG. 4). Some of these molecules, e.g., SAM andSAOH, even emit at ˜760 nm (FIG. 5), which can potentially be used fornear infra-red fluorescence imaging. In addition to fluorescencetitration, the binding of Aβ peptide and fibril with the carbazole-basedfluorophores of the present invention were further confirmed by circulardichroism spectroscopy (FIG. 6), and electrospray ionization-massspectrometry (ESI-MS). Total Internal Reflection Fluorescence Microscope(TIRFM) technique developed by us was used to investigate the inhibitioneffects of these functional fluorophores on Aβ fibril formation (FIG.7). Remarkably, some of these molecules, e.g., SLOH, SLE, SLOH-Pr,Me-SLM, SAM, and SAOH, were found to inhibit Aβ peptide aggregation andprevent fibril growth (FIG. 7). Such inhibitory effect was furtherconfirmed by Transmission Electron Microscopy (TEM) study (FIG. 8).

The inhibitory effect of the carbazole-based fluorophores of the presentinvention on Aβ fibril growth was further investigated by measuring the(average) length of the Aβ fibrils formed after incubation of the Aβmonomers for 60 min with additions of SLOH at different time pointsduring this period (FIG. 9). Parallel experiments conducted without anyaddition of SLOH were used as controls. FIG. 9 shows that an addition ofSLOH to the Aβ monomer strongly arrests its fibril growth. These resultsclearly indicate that the inhibitory effect of these carbazole-basedfluorophores on Aβ aggregation is instantaneous.

To ascertain its potential clinical application, the cytotoxicities ofthese carbazole-based molecules, SLOH, SLOH-Pr, Me-SLM, and SAOH towardsthe neuronal cell, i.e., SH-SY5Y cell line, were investigated by MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reductionassay. The results obtained (FIG. 10) showed that these molecules wereessentially non-toxic (≦20%) to the neuronal cell particularly at lowdosage.

Since it is the Aβ oligomers and fibrils that are neurotoxic, furtherexperiments with these carbazole-based molecules conducted in thepresence of the Aβ monomer (non-toxic), the neurotoxic Aβ oligomers andfibrils showed that the neuronal cells became protected from theneurotoxic effects of the Aβ oligomers and fibrils when incubated withcarbazole-based molecules SLOH and SAOH for 2 and 6 hours (FIG. 11).

However, in order for the observed neuroprotective effect to beclinically useful, these molecules need to be able to pass through theblood-brain barrier. The ability of these molecules to penetrate theblood-brain barrier was demonstrated in transgenic mice (FIG. 12). Inaddition, FIG. 12 d-f shows the selectivity of SLOH towards Aβ plaquesas confirmed with Aβ antibody which was used to identify the Aβ plaquesin transgenic mice's brain.

In summary, carbazole-based fluorophores of the present invention havebeen shown to bind to Aβ₍₁₋₄₀₎ and Aβ₍₁₋₄₂₎ as well as Aβ aggregateswith dramatic fluorescence enhancement, thus allowing their directimaging and labeling as well as the use of TIRFM technique to study theeffects of these molecules on Aβ aggregation/fibrillation. Someembodiments of the carbazole-based fluorophores, for instance, SLOH andSAOH, have been shown to be a potent inhibitor of Aβ aggregation,non-toxic and exhibiting a protective effect against the neurotoxicactivities of the Aβ oligomers and fibrils towards neuronal cells. Theseproperties, together with the ability to cross the blood-brain barrierand target the Aβ plaques, render the fluorophores of the presentinvention a potential neuroprotective and, perhaps, therapeutic agentfor Alzheimer's disease.

The following compositions according to the invention were prepared andexemplified as shown in FIG. 13. By adapting the convergent approachestablished previously, the Knoevenagel reaction ofcarbazolyl-3-aldehyde and the corresponding 4-methylpyridium or4-methylquinolinium halide was used as the key step to synthesizevarious carbazole-based cyanines. Alkylation of carbazole with ethyleneglycol chloride and methyl iodide in the presence of NaH in DMF gavealkylated carbazole 1a and 1b respectively. Monobromination of 1a and 1bin the presence of NBS gave alkylated 3-bromocarbazole, 2a and 2b,respectively. Formylation of 2a and 2b via lithiation bromide exchangeat low temperature followed by the subsequent quenching with DMFafforded carbazolyl-3-aldehyde, 3a and 3b, respectively, in moderateyield. Alkylation of lepidine or picoline was carried out in methanol oracetonitrile affording the corresponding halide, 4-9 in good to highyield. The Knoevenagel reaction of aldehyde 3a or 3b and thecorresponding 4-methylpyridium or 4-methylquinolinium halide in thepresence of piperidine in ethanol afforded the correspondingcarbazole-based cyanines in a moderate yield. For the acridine-basedcyanines dyes, 9-methylacridine was first brominated with NBS affordingbrominated product 10, which gave phosphonate ester 11 by refluxing withtriethyl phosphite. Condensation of phosphonate 11 and aldehyde 3a inthe presence of NaH afforded 12, which was alkylated with methyl iodideand 2-iodoethanol to give SAM and SAOH, respectively. All the cyanineswere fully characterized with spectroscopic techniques and found to bein good agreement with its structure.

All the solvents were dried by the standard methods wherever needed. ¹HNMR spectra were recorded using a Bruker-400 NMR spectrometer andreferenced to the residue CHCl₃ 7.26 ppm or DMSO-d₆ 2.5 ppm. ¹³C NMRspectra were recorded using a Bruker-400 NMR spectrometer and referencedto the CDCl₃ 77 ppm or DMSO-d₆ 39.5 ppm. Mass Spectroscopy (MS)measurements were carried out by using fast atom bombardment on the APIASTER Pulser I Hybrid Mass Spectrometer or matrix-assisted laserdesorption ionization-time-of-flight (MALDI-TOF) technique. Elementalanalysis was carried on the CARLO ERBA 1106 Elemental Analyzer. Compound8 and SPM were synthesized according to previous procedure.

Although the cause and progression of AD are not well understood yet,early detection and diagnosis allows preventive and delaying measuresfor the progression to AD. Thus, the development of a powerful imagingtechnique with sensitivity at the molecular level for AD diagnosis iscrucial to assess the disease status as well as the evaluation ofeffectiveness of potential AD drugs. Various imaging techniquesincluding magnetic resonance imaging, positron emission tomography,near-infrared fluorescence imaging and multiphoton excited imaging havebeen explored for amyloid plaques imaging. All these techniques requirea functional probe that can selectively target the Aβ species.

Apart from the use in direct imaging or labeling of Aβ aggregates, thecarbazole-based fluorophores of the present invention is also useful asa magnetic resonance imaging (MRI) contrast agent that bind beta amyloidpeptides. By conjugating appropriate paramagnetic metal complexes tothese carbazole-based fluorophores, these compounds can potentially bedeveloped into beta-amyloid peptide-specific MRI contrast agents. Toconvert these Aβ fibril-specific carbazole-based fluorophores dyes intoMRI contrast agents, we can attach strongly paramagnetic and kineticallyinert metal complexes, such as the gadolinium(III), iron(III), andmanganese(II) complexes, via the R₁ side chain of the carbazole moietyto these fluorophores. Gd(III)-based chelates, such as [Gd(DTPA)(H₂O)]²⁻(DTPA=diethylenetriaminepentaacetic acid), approved for clinical use in1988 and commercially known as Magnevist, are attractive candidates.Recently, further enhancement of the MRI contrast properties of theseGd(III) complexes was achieved by allowing the coordination of a secondinner-sphere water molecule, which raised the relaxivity of theconventional Gd(III) contrast agents from 4-5 mM⁻¹ s⁻¹ (at 20 MHz fieldstrength) to 10.5 mM⁻¹ s⁻¹, in the Gd-TREN-1-Me-3,2-HOPO complex, [1](where TREN=tris(2-aminoethyl), HOPO=hydroxypyridinone, structure shownbelow).

A slight modification of one of the hydroxypyridinone ligands of theGd(III) complex, shown in [2], allows flexible attachment to thecarbazole moiety of A fibril-specific dyes via, for example, apolyethylene glycol (PEG) linkage.

More recently, a series of ¹H/¹⁹F dual MR imaging agents based onCF₃-labeled lanthanide(III) complexes (Ln=Gd, Tb, Dy, Ho, Er, Tm) withamide-substituted 1,4,7,10-tetraazacyclododecane ligand have beendesigned. An example of this ligand system bearing a CF₃ reporter groupis shown in [3].

The advantage of ¹⁹F MRI is the exquisite sensitivity of the ¹⁹F shiftof the reporter group to its local chemical environment, thus opening upthe possibility of responsive MRI to detect changes in local pH, oxygenstress, etc. The fact that standard MRI instruments can be easily tunedfrom ¹H to ¹⁹F nuclei, which have very similar magnetic properties, isan added bonus of this technique. This ligand system is also amenable tocoupling (e.g., at the −X or −Y positions indicated) to the carbazolemoiety of the carbazole-based fluorophores dyes.

SYNTHESIS EXAMPLES 9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole (1a)

To a solution of carbazole (3.34 g, 20 mmol) in DMF (80 mL) at 0° C. wasadded NaH (0.72 g, 30 mmol). After heating to 80° C. for 1.5 h,1-chloro-2-(2-methoxyethoxy)ethane (3.31 g, 24 mmol) was added dropwise.The resulting mixture was kept at 80° C. overnight. After cooling downto 0° C., the reaction mixture was carefully quenched with water andextracted with ethyl acetate three times. The combined organic phase waswashed with water and brine. Then the organic layer was dried overanhydrous sodium sulfate and the solvent was removed. The residue waspurified by silica gel chromatography using petroleum ether and ethylacetate as eluent (EA:PE=1:3) to afford alkylated carbazole 1a (4.46 g)as brown oil in 83% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.09 (d, J=7.6 Hz,2H), 7.46 (m, 4H), 7.23 (m, 2H), 4.51 (t, J=6.4 Hz, 2H), 3.86 (t, J=6.4Hz, 2H), 3.52 (m, 2H), 3.42 (m, 2H), 3.31 (s, 3H). ¹³C NMR (400 MHz,CDCl₃) δ 140.5, 125.6, 122.8, 120.2, 118.9, 108.7, 71.8, 70.7, 69.1,59.0, 43.0. MS (FAB) m/z Calcd for C₁₇H₁₉NO₂ 269.1 Found 269.2 [M]⁺.

9-methyl-9H-carbazole (1b)

To a solution of carbazole (3.34 g, 20 mmol) in DMF (80 mL) at 0° C. wasadded NaH (0.72 g, 30 mmol). After heating at 80° C. for 1.5 h,iodomethane (3.4 g, 24 mmol) was added dropwise. The resulting mixturewas kept at 80° C. overnight. After cooling down to 0° C., the reactionmixture was carefully quenched with water and extracted with ethylacetate three times. The combined organic phase was washed with waterand brine. Then the organic layer was dried over anhydrous sodiumsulfate and the solvent was removed. The residue was purified by silicagel chromatography using petroleum ether and ethyl acetate as eluent(EA:PE=1:5) to afford methylated carbazole 1b (2.78 g) as yellow oil in77% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.08 (d, J=8.0 Hz, 2H), 7.46 (t,J=8.0 Hz, 2H), 7.36 (d, J=8.0 Hz, 2H), 7.22 (t, J=8.0 Hz, 2H), 3.79 (s,3H).¹³C NMR (400 MHz, CDCl₃) δ 140.9, 125.6, 122.7, 120.2, 118.8, 108.4,28.9.

3-bromo-9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole (2a)

To a solution of 1a (2 g, 7.4 mmol) in dichloromethane (60 mL) was addedNBS (1.3 g, 7.4 mmol) portionwise in an ice-water bath. After completeaddition, the solution mixture was warmed to room temperature andstirred overnight. The resulting solution was washed with water andbrine. The organic phase was dried over anhydrous sodium sulfate and thesolvent was removed. The residue was purified by silica gelchromatography using ethyl acetate and petroleum ether (EA:PE=1:5) aseluent to afford 2a (1.75 g) in 68% yield as an oil that can turn intosolid after standing. ¹H NMR (400 MHz, CDCl₃) δ 8.16 (d, J=2.0 Hz, 1H),8.01 (d, J=8.0 Hz, 1H), 7.51 (dd, J=8.0 Hz, 2.0 Hz, 1H), 7.44 (m, 2H),7.34 (d, J=8.4 Hz, 1H), 7.22 (m, 1H), 4.46 (t, J=6.0 Hz, 2H), 3.83 (t,J=6.0 Hz, 2H), 3.48 (m, 2H), 3.39 (m, 2H), 3.28 (s, 3H). ¹³C NMR (400MHz, CDCl₃) δ 140.7, 139.2, 128.2, 126.3, 124.5, 122.8, 121.8, 120.4,119.3, 111.7, 110.4, 109.0, 71.8, 70.7, 69.1, 59.0, 43.2. MS (FAB) m/zCalcd for C₁₇H₁₈BrNO₂ 347.0 Found 347.3 [M]⁺.

3-bromo-9-methyl-9H-carbazole (2b)

To a solution of 1b (2.5 g, 13.8 mmol) in dichloromethane (80 mL) wasadded NBS (2.4 g, 13.8 mmol) portion-wise in an ice-water bath. Aftercomplete addition, the solution mixture was warmed to room temperatureand stirred overnight. The resulting solution was washed with water andbrine. The organic phase was dried over anhydrous sodium sulfate and thesolvent was removed. The residue was purified by silica gelchromatography using ethyl acetate and petroleum ether (EA:PE=1:10) aseluent to afford 2b (2.11 g) in 59% yield. ¹H NMR (400 MHz, CDCl₃) δ8.19 (d, J=2.0 Hz, 1H), 8.03 (d, J=8.0 Hz, 1H), 7.54 (dd, J=8.8 Hz,J=2.0 Hz, 1H), 7.50 (td, J=8.0 Hz, J=1.2 Hz, 1H), 7.39 (d, J=8.0 Hz,1H), 7.27-7.22 (m, 2H), 3.82 (s, 3H).

9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole-3-carbaldehyde (3a)

To a solution of 2a (1.5 g, 4.3 mmol) in dried THF (45 mL) was addedn-BuLi (3.5 mL 5.2 mmol) at −78° C. The resulting mixture was stirred at−78° C. for 1 h and then added with dried DMF (3 mL). The reactionmixture was allowed to warm to room temperature and stirred overnightbefore quenched with aqueous ammonia chloride solution. Water was addedand extracted with ethyl acetate three times. The combined organic phasewas washed with brine and dried over anhydrous sodium sulfate. Afterremoving the solvent, the residue was purified by silica gelchromatography using ethyl acetate and petroleum ether (EA:PE=1:2) aseluent to afford 3a (0.76 g) as yellow solid in 60% yield. ¹H NMR (400MHz, CDCl₃) δ 10.07 (s, 1H), 8.58 (d, J=0.8 Hz, 1H), 8.13 (d, J=8.0 Hz,1H), 7.98 (dd, J=8.8 Hz, 0.8 Hz, 1H), 7.51 (m, 3H), 7.30 (m, 1H), 4.53(t, J=6.0 Hz, 2H), 3.87 (t, J=6.0 Hz, 2H), 3.49 (m, 2H), 3.38 (m, 2H),3.26 (s, 3H). ¹³C NMR (400 MHz, CDCl₃) δ 191.8, 144.3, 141.1, 128.5,127.1, 126.6, 123.7, 123.0, 122.9, 120.6, 120.4, 109.4, 109.3, 71.8,70.8, 69.1, 59.0, 43.4. MS (FAB) m/z Calcd for C₁₈H₁₉NO₃ 297.1 Found297.3 [M]⁺.

9-methyl-9H-carbazole-3-carbaldehyde (3b)

To a solution of 2b (1.8 g, 6.9 mmol) in dried THF (45 mL) was addedn-BuLi (3.3 mL 8.3 mmol) at −78° C. The resulting mixture was stirred at−78° C. for 1 h and then added with dried DMF (8 mL). The reactionmixture was allowed to warm to room temperature and stirred overnightbefore quenched with aqueous ammonia chloride solution. Water was addedand extracted with ethyl acetate three times. The combined organic phasewas washed with brine and dried over anhydrous sodium sulfate. Afterremoving the solvent, the residue was purified by silica gelchromatography using ethyl acetate and petroleum ether (EA:PE=1:4) aseluent to afford 3b (0.86 g) in 60% yield. ¹H NMR (400 MHz, CDCl₃) δ9.58 (s, 1H), 7.79 (s, 1H), 7.49 (d, J=7.6 Hz, 1H), 7.41 (d, J=8.8 Hz,1H), 7.09 (t, J=7.6 Hz,), 6.90 (t, J=7.6 Hz, 1H), 6.77 (d, J=8.0 Hz,1H), 6.61 (d, J=8.4 Hz, 1H), 3.00 (s, 3H). ¹³C NMR (400 MHz, CDCl₃) δ190.9, 143.2, 140.5, 127.4, 125.8, 122.7, 121.7, 119.6, 119.4, 108.3,107.6, 28.0.

1,4-dimethylquinolinium iodide (4)

A solution mixture of lepidine (0.66 g, 4.65 mmol) and iodomethane (1.32g, 9.3 mmol) in methanol (30 mL) was heated to reflux in a sealed tubeovernight. After cooling to room temperature, methanol was removed undervacuum. Anhydrous acetone was added to the residue and filtered. Theresulting solid was washed with acetone and dried to afford iodide 4(1.1 g) as yellow solid in 83% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 9.35(d, J=6 Hz, 1H), 8.54 (d, J=8.8 Hz, 1H), 8.49 (d, J=8.8 Hz, 1H), 8.27(t, J=7.2 Hz, 1H), 8.07 (t, J=4.8 Hz, 1H), 8.05 (d, J=6 Hz, 1H), 4.57(s, 3H), 3.00 (s, 3H). ¹³C NMR (400 MHz, DMSO-d₆) δ 158.1, 148.9, 137.6,134.9, 129.6, 128.4, 126.8, 122.4, 119.5, 44.9, 19.6. MS (FAB) m/z Calcdfor C₁₁H₁₂N^(+158.0) Found 158.2 [M]⁺.

1-(2-hydroxyethyl)-4-methylquinolinium chloride (5)

A solution mixture of lepidine (0.8 g, 5.6 mmol) and 2-chloroethanol(2.25 g, 28 mmol) in acetonitrile (15 mL) was heated to 120° C. in asealed tube overnight. After cooling to room temperature, the solventwas removed. The resulting mixture was precipitate from methanol andethyl acetate to give the desired product 5 (0.79 g) in 63% yield. ¹HNMR (400 MHz, DMSO-d₆) δ 9.24 (d, J=6 Hz, 1H), 8.61 (d, J=7.2 Hz, 1H),8.55 (d, J=7.2 Hz, 1H), 8.25 (m, 1H), 8.06 (m, 2H), 5.15 (br, 1H), 5.08(t, J=4.8 Hz, 2H), 3.91 (t, J=4.8 Hz, 2H), 3.01 (s, 3H). ¹³C NMR (400MHz, DMSO-d₆) δ 158.8, 149.2, 137.1, 135.1, 129.7, 129.1, 127.2, 122.4,119.5, 59.4, 59.0, 19.9. MS (FAB) m/z Calcd for C₁₂H₁₄NO^(+188.2) Found188.2 [M]⁺.

1-ethyl-4-methylquinolinium bromide (6)

A solution mixture of lepidine (0.5 g, 3.5 mmol) and bromoethane (1.96g, 18 mmol) in acetonitrile (15 mL) was heated to reflux overnight.After cooling to room temperature, the solvent was removed. Theresulting mixture was precipitate from methanol and ethyl acetate togive the desired product 6 (0.81 g) in 92% yield. ¹H NMR (400 MHz,DMSO-d₆) δ 9.44 (d, J=6 Hz, 1H), 8.60 (d, J=9.2 Hz, 1H), 8.54 (dd, J=8.4Hz, J=1.2 Hz, 1H), 8.26 (td, J=8.0 Hz, J=1.6 Hz, 1H), 8.09-8.04 (m, 2H),5.06 (tr, J=7.2 Hz, 2H), 3.00 (s, 3H), 1.58 (t, J=7.2 Hz, 3H). ¹³C NMR(400 MHz, DMSO-d₆) δ 158.4, 148.2, 136.6, 135.1, 129.6, 128.9, 127.2,122.8, 119.2, 52.5, 19.7, 15.2.

1-(3-hydroxypropyl)-4-methylquinolinium bromide (7)

A solution mixture of lepidine (0.5 g, 3.5 mmol) and 3-bromopropanol(1.9 g, 14 mmol) in acetonitrile (15 mL) was heated to reflux overnight.After cooling to room temperature, the solvent was removed. Theresulting mixture was precipitate from methanol and ethyl acetate togive the desired product 7 (0.83 g) in 84% yield. ¹H NMR (400 MHz,DMSO-d₆) δ 9.41 (d, J=6 Hz, 1H), 8.58 (d, J=8.8 Hz, 1H), 8.54 (dd, J=8.8Hz, J=1.2 Hz, 1H), 8.26 (td, J=8.0 Hz, J=1.2 Hz, 1H), 8.08-8.03 (m, 2H),5.09 (t, J=6.8 Hz, 2H), 3.52 (t, J=5.6 Hz, 2H), 3.01 (s, 3H), 2.15-2.08(m, 2H). ¹³C NMR (400 MHz, DMSO-d₆) δ 158.5, 148.8, 136.8, 135.1, 129.5,128.9, 127.2, 122.6, 119.3, 57.4, 54.8, 32.0, 19.7.

1-(2-hydroxyethyl)-4-methylpyridinium chloride (9)

A solution mixture of picoline (0.93 g, 10 mmol) and 2-chloroethanol(4.03 g, 50 mmol) in acetonitrile (20 mL) was heated to 120° C. in asealed tube overnight. After cooling to room temperature, the solventwas removed under vacuum. The resulting mixture was precipitate frommethanol and ethyl acetate to give the desired product 9 (1.5 g) in 87%yield. ¹H NMR (400 MHz, DMSO-d₆) δ 8.94 (d, J=6.4 Hz, 2H), 7.98 (d,J=6.4 Hz, 2H), 5.55 (br, 1H), 4.64 (t, J=4.8 Hz, 2H), 3.81 (t, J=4.8 Hz,2H), 2.60 (s, 3H).

¹³C NMR (400 MHz, DMSO-d₆) δ 158.7, 144.2, 127.9, 62.1, 60.0, 21.4.

(E)-1-(2-hydroxyethyl)-4-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)pyridiniumchloride (SPOH)

A solution mixture of 3a (0.13 g, 0.75 mmol), 9 (0.27 g, 0.9 mmol) andpiperidine (0.1 mL) in ethanol (30 mL) was heated to reflux overnight.After cooling down to room temperature, the organic solvent was removedby rotary evaporation. The residue was purified by recrystallizationfrom methanol affording SPOH (0.18 g) as pale red solid in 53% yield. ¹HNMR (400 MHz, DMSO-d₆) δ 8.88 (d, J=6.8 Hz, 2H), 8.55 (s, 1H), 8.19 (m,4H), 7.84 (d, J=8 Hz, 1H), 7.65 (m, 2H), 7.49 (m, 2H), 7.25 (t, J=7.2Hz, 1H), 5.66 (s, 1H), 4.57 (m, 4H), 3.79 (m, 4H), 3.43 (m, 2H), 3.27(m, 2H), 3.08 (s, 3H). ¹³C NMR (400 MHz, DMSO-d₆) δ 153.4, 144.4, 142.4,141.7, 140.8, 126.4, 126.3, 126.2, 122.7, 122.6, 122.1, 121.1, 120.3,120.0, 119.7, 110.4, 110.2, 71.2, 69.8, 68.8, 61.6, 600.1, 58.1, 42.8.HRMS (MALDI-TOF) m/z Calcd for C₂₆H₂₉N₂O₃ 417.2172 Found 417.2184 [M⁺].

(E)-4-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)-1-methylquinoliniumiodide (SLM)

A solution mixture of 3a (0.14 g, 0.5 mmol), 4 (0.18 g, 0.6 mmol) andpiperidine (0.1 mL) in ethanol (40 mL) was heated to reflux overnight.After cooling down to room temperature, the organic solvent was removed.The residue was purified by recrystallization from methanol to affordSLM (0.24 g) as red solid in 56% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 9.28(d, J=6.4 Hz, 1H), 9.14 (d, J=8.4 Hz, 1H), 8.86 (s, 1H), 8.51 (d, J=6.4Hz, 1H), 8.42 (m, 3H), 8.28 (m, 2H), 8.13 (d, J=8.8 Hz, 1H), 8.08 (t,J=7.2 Hz, 1H), 7.80 (d, J=8.8 Hz, 1H), 7.71 (d, J=8.0 Hz, 1H), 7.53 (t,J=8.0 Hz, 1H), 7.32 (t, J=7.2 Hz, 1H), 4.64 (t, J=5.2 Hz, 2H), 4.52 (s,3H), 3.84 (t, J=5.2 Hz, 2H), 3.48 (m, 2H), 3.33 (m, 2H), 3.11 (s, 3H).¹³C NMR (400 MHz, DMSO-d₆) δ 153.0, 147., 144.9, 142.1, 140.9, 138.8,134.9, 129.0, 127.3, 126.7, 126.4, 126.1, 122.8, 122.2, 121.7, 120.4,119.9, 119.3, 116.2, 115.1, 110.5, 110.4, 71.3, 69.8, 68.9, 58.1, 44.2,42.9. HRMS (MALDI-TOF) m/z Calcd for C₂₉H₂₉N₂O₂ 437.2223 Found 437.2207[M⁺].

(E)-1-(2-hydroxyethyl)-4-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)-quinoliniumchloride (SLOH)

A solution mixture of 3a (0.12 g, 0.55 mmol), 5 (0.2 g, 0.66 mmol) andpiperidine (0.1 mL) in ethanol (35 mL) was heated to reflux overnight.After cooling down to room temperature, the organic solvent was removed.The residue was purified by recrystallization from methanol to affordSLOH (0.17 g) as red solid in 62% yield. ¹H NMR (400 MHz, DMSO-d₆) δ9.20 (d, J=6.4 Hz, 1H), 9.15 (d, J=8.8 Hz, 1H), 8.87 (s, 1H), 8.56 (d,J=9.2 Hz, 1H), 8.52 (d, J=6.4 Hz, 1H), 8.40 (m, 2H), 8.24 (m, 2H), 8.13(d, J=8.8 Hz, 1H), 8.05 (t, J=7.6 Hz, 1H), 7.78 (d, J=8.8 Hz, 1H), 7.71(d, J=8.4 Hz, 1H), 7.52 (t, J=8.0 Hz, 1H), 7.31 (t, J=7.6 Hz, 1H), 5.27(t, J=5.6 Hz, 1H), 5.05 (t, J=4.8 Hz, 2H), 4.64 (t, J=4.8 Hz, 2H), 3.94(m, 2H), 3.84 (t, J=5.2 Hz, 2H), 3.47 (m, 2H), 3.31 (m, 2H), 3.11 (s,3H). ¹³C NMR (400 MHz, DMSO-d₆) δ 153.3, 147.8, 145.0, 142.1, 140.9,138.1, 134.7, 128.7, 127.1, 126.8, 126.7, 126.5, 122.8, 122.2, 121.7,120.3, 119.8, 119.2, 116.3, 114.8, 110.4, 110.3, 71.2, 69.8, 68.8, 58.9,58.5, 58.0, 42.9. HRMS (MALDI-TOF) m/z Calcd for C₃₀H₃₁N₂O₃ 467.2342Found 467.2340 [M⁺]. Calcd for C₃₀H₃₁ClN₂O₃: C, 71.53; H, 6.21; N, 5.57.Found: C, 71.04; H, 6.23; N, 5.36.

(E)-1-ethyl-4-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)quinoliniumbromide (SLE)

A solution mixture of 6 (0.20 g, 0.8 mmol), 3a (0.33 g, 1.1 mmol) andpiperidine (0.1 mL) in ethanol (40 mL) was heated to reflux overnight.After cooling down to room temperature, the organic solvent was removed.The residue was purified by precipitation from methanol and ethylacetate to afford SLE (0.22 g) in 53% yield.¹H NMR (400 MHz, DMSO-d₆) δ9.34 (d, J=8.4 Hz, 1H), 9.15 (d, J=8.4 Hz, 1H), 8.86 (s, 1H), 8.54-8.51(m, 2H), 8.44 (d, J=16 Hz, 1H), 8.36 (d, J=16 Hz, 1H), 8.28-8.23 (m,2H), 8.12 (d, J=8.0 Hz, 1H), 8.05 (t, J=7.6 Hz, 1H), 7.77 (d, J=8.4 Hz,1H), 7.70 (d, J=8.4 Hz, 1H), 7.52 (t, J=7.6 Hz, 1H), 7.31 (t, J=7.6 Hz,1H), 4.99 (tr, J=6.8 Hz, 2H), 4.63 (t, J=4.8 Hz, 2H), 3.84 (t, J=4.8 Hz,2H), 3.48 (t, J=4.8 Hz, 2H), 3.31 (t, J=4.8 Hz, 2H), 3.11 (s, 3H), 1.59(t, J=6.8 Hz, 3H). ¹³C NMR (400 MHz, DMSO-d₆) δ 153.2, 146.7, 145.1,142.2, 140.9, 137.7, 135.0, 128.9, 127.4, 126.8, 126.7, 126.5, 126.4,122.8, 122.2, 121.8, 120.4, 119.9, 119.0, 116.2, 115.5, 110.4, 110.3,71.3, 69.8, 68.9, 58.1, 51.9, 15.1. HRMS (MALDI-TOF) m/z Calcd forC₃₀H₃₁N₂O₂ 451.2380 Found 451.2362 [M]⁺.

(E)-1-(3-hydroxypropyl)-4-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)-quinoliniumbromide (SLOH-Pr)

A solution mixture of 7 (0.17 g, 0.6 mmol), 3a (0.24 g, 0.8 mmol) andpiperidine (0.1 mL) in ethanol (40 mL) was heated to reflux overnight.After cooling down to room temperature, the organic solvent was removed.The residue was purified by precipitation from methanol and ethylacetate to afford SLOH-Pr (0.14 g) in 41% yield.¹H NMR (400 MHz,DMSO-d₆) δ 9.29 (d, J=6.8 Hz, 1H), 9.15 (d, J=8.4 Hz, 1H), 8.88 (s, 1H),8.51 (d, J=6.8 Hz, 1H), 8.45 (d, J=16 Hz, 1H), 8.37 (d, J=16 Hz, 1H),8.28-8.24 (m, 2H), 8.13 (d, J=8.4 Hz, 1H), 8.05 (t, J=8.0 Hz, 1H), 7.77(d, J=8.8 Hz, 1H), 7.70 (d, J=8.4 Hz, 1H), 7.52 (t, J=7.6 Hz, 1H), 7.31(t, J=7.2 Hz, 1H), 5.01 (t, J=7.2 Hz, 2H), 4.86 (t, J=5.2 Hz, 1H), 4.63(t, J=4.8 Hz, 2H), 3.84 (t, J=5.2 Hz, 2H), 3.55 (tr, J=5.2 Hz, 2H), 3.47(t, J=5.6 Hz, 2H), 3.31 (t, J=4.8 Hz, 2H), 3.11 (s, 3H), 2.13 (t, J=6.0Hz, 2H). ¹³C NMR (400 MHz, DMSO-d₆) δ 153.3, 147.3, 145.1, 142.2, 140.9,137.9, 135.0, 128.8, 127.4, 126.8, 126.7, 126.5, 126.4, 122.8, 122.2,121.8, 120.4, 119.9, 119.0, 116.3, 115.2, 110.5, 110.4, 71.3, 69.8,68.9, 58.1, 57.6, 54.2, 42.9, 32.0. HRMS (MALDI-TOF) m/z Calcd forC₃₁H₃₃N₂O₃ 481.2485 Found 481.2458 [M]⁺.

(E)-1-methyl-4-(2-(9-methyl-9H-carbazol-3-yl)vinyl)quinolinium iodide(Me-SLM)

A solution mixture of 1,4-dimethylquinolinium iodide (0.14 g, 0.5 mmol),3b (0.13 g, 0.6 mmol) and piperidine (0.1 mL) in ethanol (40 mL) washeated to reflux overnight. After cooling down to room temperature, theorganic solvent was removed. The residue was purified by precipitationfrom methanol and ethyl acetate to afford Me-SLM (0.14 g) in 62% yield.¹H NMR (400 MHz, DMSO-d₆) δ 9.27 (d, J=6.4 Hz, 1H), 9.12 (d, J=8.4 Hz,1H), 8.86 (s, 1H), 8.49 (d, J=6.4 Hz, 1H), 8.45-8.23 (m, 5H), 8.15 (d,J=8.8 Hz, 1H), 8.06 (t, J=7.6 Hz, 1H), 7.75 (d, J=8.4 Hz, 1H), 7.66 (d,J=8.0 Hz, 1H), 7.55 (t, J=7.6 Hz, 1H), 7.32 (t, J=7.6 Hz, 1H), 4.51 (s,3H), 3.95 (s, 3H).¹³C NMR (400 MHz, DMSO-d₆) δ 152.9, 147.3, 144.8,142.2, 141.2, 138.7, 134.8, 128.8, 127.4, 126.6, 126.4, 126.3, 126.0,122.6, 122.0, 121.8, 120.4, 119.7, 119.1, 116.0, 115.0, 109.8, 109.7,44.3, 29.3. HRMS (MALDI-TOF) m/z Calcd for C₂₅H₂₁N₂ 349.1699 Found349.1694 [M]⁺.

9-(bromomethyl)acridine (10)

To a solution of 9-methylacridine (1.93 g, 10 mmol) in dichloromethane(100 mL) was added NBS (1.78 g, 10 mmol) portion-wise in an ice-waterbath. After complete addition, the solution mixture was warmed to roomtemperature and stirred overnight. The resulting solution was washedwith water and brine. The organic phase was dried over anhydrous sodiumsulfate and the solvent was removed. The residue was purified by silicagel chromatography using ethyl acetate and petroleum ether (EA:PE=1:5)as eluent to afford 10 (2.08 g) in 77% yield. ¹H NMR (400 MHz, CDCl₃) δ8.27 (d, J=8.8 Hz, 4H), 7.81 (t, J=8.0 Hz, 2H), 7.68 (t, J=8.0 Hz, 2H),5.42 (s, 2H). ¹³C NMR (400 MHz, CDCl₃) δ 148.9, 138.7, 130.5, 130.1,126.8, 123.8, 123.4, 23.1. MS (FAB) m/z Calcd for C₁₄H₁₀BrN 272.1 Found2722. [M]⁺.

Diethyl acridin-9-ylmethylphosphonate (11)

The mixture of 10 (1.5 g, 5.5 mmol) and triethyl phosphite (2 mL) washeated to reflux for 4 h. After cooling down to room temperature, theexcess triethyl phosphite was removed under vacuum to afford 11 (1.7 g)in 94% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.23 (d, J=8.8 Hz, 2H), 8.17 (d,J=8.8 Hz, 2H), 7.72 (t, J=7.2 Hz, 2H), 7.54 (t, J=7.2 Hz, 2H), 4.13 (d,J=24 Hz, 2H), 3.92-3.77 (m, 4H), 1.04 (t, J=7.2 Hz, 6H). ¹³C NMR (400MHz, CDCl₃) δ 148.4, 148.3, 135.8, 135.7, 129.9, 129.8, 125.8, 125.3,125.2, 124.9, 124.8, 62.4, 27.5, 26.1, 16.1.

(E)-9-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)acridine(12)

To a solution of 3a (0.45 g, 1.5 mmol) and 11 (0.49 g, 1.5 mmol) in dryTHF (45 mL), NaH (45 mg, 1.8 mmol) was added carefully in an ice-waterbath. After complete addition, the solution mixture was warmed to roomtemperature and stirred overnight. After quenching by water, theresulting mixture was extracted with ethyl acetate for three times. Thecombined organic phase was washed with brine twice and dried overanhydrous sodium sulfate. After removing the solvent, the resultingcrude product was purified by silica gel chromatography using DCM andpetroleum ether (DCM:PE=1:10) to afford 12 (0.45 g) in 64% yield. ¹H NMR(400 MHz, CDCl₃) δ 8.45 (d, J=8.8 Hz, 2H), 8.37 (s, 1H), 8.26 (d, J=8.8Hz, 2H), 8.15 (d, J=8.0 Hz, 1H), 7.95 (d, J=8.4 Hz, 1H), 7.85 (d, J=8.8Hz, 1H), 7.80 (t, J=8.0 Hz, 2H), 7.58-7.51 (m, 5H), 7.31-7.25 (m, 2H),4.58 (t, J=6.4 Hz, 2H), 3.92 (t, J=6.4 Hz, 2H), 3.57-3.55 (m, 2H),3.48-3.45 (m, 2H), 3.35 (s, 3H). ¹³C NMR (400 MHz, CDCl₃) δ 148.9,143.8, 141.0, 140.6, 129.9, 127.9, 126.1, 125.4, 124.6, 123.4, 122.9,120.4, 119.5, 119.2, 119.1, 109.4, 109.2, 71.9, 70.9, 69.3, 59.1, 43.3.HRMS (MALDI-TOF) m/z Calcd for C₃₂H₂₉N₂O₂ 473.2223 Found 473.2210[M+H]⁺.

(E)-9-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)-10-methylacridiniumiodide (SAM)

A solution of 12 (0.20 g, 0.4 mmol) and methyl iodide (0.57 g, 4 mmol)in acetonitrile (8 mL) was heated to 100° C. in sealed tube for 24 h.After cooling down to room temperature, the solvent was removed and theresulting mixture was purified by precipitation from methanol and ethylacetate to afford SAM (0.15 g) in 61% yield. ¹H NMR (400 MHz, CDCl₃) δ8.74 (d, J=8.0 Hz, 2H), 8.49 (s, 1H), 8.46 (d, J=8.8 Hz, 2H), 8.31 (d,J=16 Hz, 1H), 8.26 (t, J=8.0 Hz, 2H), 8.10 (d, J=8.0 Hz, 1H), 7.93 (d,J=8.0 Hz, 1H), 7.83 (t, J=7.2 Hz, 2H), 7.51 (d, J=8.4 Hz, 1H), 7.46 (t,J=6.4 Hz, 2H), 7.44 (d, J=16 Hz, 1H), 7.20 (t, J=6.4 Hz, 1H), 4.82 (s,3H), 4.46 (t, J=6.0 Hz, 2H), 3.88 (t, J=6.0 Hz, 2H), 3.55-3.53 (m, 2H),3.44-3.42 (m, 2H), 3.30 (s, 3H). ¹³C NMR (400 MHz, CDCl₃) δ 157.9,149.5, 141.8, 140.5, 140.1, 137.8, 129.3, 127.4, 126.9, 126.5, 126.2,123.9, 123.2, 122.1, 121.2, 121.1, 119.9, 117.9, 117.4, 109.4, 109.0,71.7, 70.6, 69.0, 58.9, 43.3, 39.5. HRMS (MALDI-TOF) m/z Calcd forC₃₃H₃₁N₂O₂ ^(+487.2380) Found 487.2387 [M]⁺.

(E)-10-(2-hydroxyethyl)-9-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)-acridiniumiodide (SAOH)

A solution of 12 (0.2 g, 0.4 mmol) and 2-iodoethanol (0.7 g, 4 mmol) inacetonitrile (10 mL) was heated to 120° C. in sealed tube for 24 h.After cooling down to room temperature, the solvent was removed and theresulting mixture was purified by precipitation from methanol and ethylacetate to afford SAOH (0.13 g) in 52% yield. ¹H NMR (400 MHz, CDCl₃) δ8.98 (d, J=9.2 Hz, 2H), 8.77 (d, J=8.4 Hz, 2H), 8.44 (s, 1H), 8.36 (t,J=8.0 Hz, 2H), 8.18 (d, J=8.4 Hz, 1H), 8.17 (d, J=16 Hz, 1H), 7.91 (d,J=8.0 Hz, 1H), 7.86 (t, J=8.0 Hz, 2H), 7.62 (d, J=8.8 Hz, 1H), 7.55 (d,J=8.4 Hz, 1H), 7.52 (t, J=6.4 Hz, 2H), 7.48 (d, J=16 Hz, 1H), 7.33 (t,J=6.4 Hz, 1H), 5.63 (t, J=6.0 Hz, 2H), 4.75 (t, J=7.6 Hz, 1H), 4.59 (t,J=6.0 Hz, 2H), 4.51-4.47 (m, 2H), 3.94 (t, J=6.0 Hz, 2H), 3.57-3.55 (m,2H), 3.47-3.44 (m, 2H), 3.33 (s, 3H). ¹³C NMR (400 MHz, CDCl₃) δ 158.2,149.1, 141.9, 140.7, 140.6, 138.1, 129.1, 126.9, 126.5, 126.4, 124.3,123.3, 122.4, 121.2, 120.9, 120.0, 119.0, 117.0, 109.6, 109.2, 71.8,70.6, 69.1, 59.3, 58.9, 52.2, 43.3. HRMS (MALDI-TOF) m/z Calcd forC₃₄H₃₃N₂O₃ ^(+517.2486) Found 517.2476 [M]⁺.

Another general chemical structures of carbazole-based fluorophoresrepresentation, including S series are shown in FIG. 14.

In FIG. 14, Ar is a heteraromatic ring selected from the groupconsisting of pyridinyl, substituted pyridinyl, quinolinyl, substitutedquinolinyl, acridinyl, substituted acridinyl, benzothiazolyl,substituted benzothiazolyl, benzoxazolyl, and substituted benzoxazolyl;

-   -   R₁ is a radical selected from the group consisting of        polyethylene glycol chain, alkyl, substituted alkyl, peptide        chain, glycosidyl, and C(O)NHCH((CH₂CH₂O)₂CH₃)₂;    -   R₂ is selected from the group consisting of ethenyl, ethynyl,        azo and azomethinyl.    -   R₃ is a radical selected from the group consisting of HO-alkyl,        alkyl-COOalkyl, alkyl-CONH₂, alkyl-CONHalkyl, polyethylene        glycol chain;    -   X is an anion selected from the group consisting of F, Cl, Br,        I, HSO₄, H₂PO₄, HCO₃, tosylate, and mesylate;    -   Y is selected from the group consisting of H. F, Cl, OH, and        OCH₃.

A novel series of water-soluble carbazole-based fluorophores aredisclosed in the present application. These molecules bind to Aβ (1-40)and Aβ (1-42) peptides and, more importantly, their oligomers, andfibrils with strong fluorescence enhancement, therefore allowing directimaging and detection for the Aβ peptides, oligomers and their fibrils.(FIG. 16) Upon binding with Aβ peptides, there is an increase influorescence intensity up to 160-fold enhancement concomitant with thesubstantial blue shifts in the emission spectra of the fluorophores.Remarkably, these molecules, including F-SLOH, SLAD, SLAce, and SLGinhibit Aβ peptide aggregation and prevent fibril growth. In oneembodiment of the present invention, Aβ is a quinolinyl or substitutedquinolinyl; R₁ is a 2-(2-methoxyethoxy)ethoxy; R₂ is an ethenyl; R₃ is a2-hydroxyethyl or acetamide or acetate or 2-(2-methoxyethoxy)ethoxy; Xis a chloride or iodide and Y is a H or F which are represented by theformula “F-SLOH”, “SLAD”, “SLAce”, and “SLG”, respectively, as shown inFIG. 15.

Total Internal Reflection Fluorescence Microscope (TIRFM) techniquedeveloped to investigate the inhibition effects of these functionalfluorophores on Aβ (1-40) fibril formation (FIG. 17). Remarkably, someof these molecules, e.g., F-SLOH, SLAD, SLAce, and SLG inhibit Aβ (1-40)peptide aggregation and prevent fibril growth (FIG. 17).

To confirm its clinical application, the cytotoxicities of thesecarbazole-based molecules towards the neuronal cell, i.e., SH-SY5Y cellline, are investigated by MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reductionassay. The results obtained (FIG. 18) show that these molecules areessentially non-toxic (20%) to the neuronal cell. The cytotoxicity ofthese molecules are low with LC₅₀=5-90 μM.

There is growing evidence showing that the soluble Aβ oligomers is themost neurotoxic form, further experiments with these carbazole-basedmolecules conducted in the presence of the Aβ monomer, Aβ oligomers andfibrils show that the primary cortical cells are protected from theneurotoxic effects of the Aβ species when incubated with the cyaninedyes, F-SLOH, and SLAD (FIG. 19). The reactive oxygen species (ROS)induced by the Aβ toxicity causes much damage in AD. Remarkably, F-SLOH,and SLAD reduce the ROS induced by the Aβ species in primary corticalcells.

However, in order for the observed neuroprotective effect to beclinically useful, these molecules need to be able to pass through theblood-brain barrier. The ability of these molecules to penetrate theblood-brain barrier is demonstrated in mice (FIG. 20). The binding ofthese molecules toward Aβ plaques in the brains of the Alzheimer'sdisease animal models are also demonstrated. Impressively, F-SLOH, SLAD,SLAce, and SLG show blood-brain permeability.

Further Synthesis Experiments:

(E)-1-(2-(2-methoxyethoxy)ethyl)-4-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)quinoliniumiodide (SLG)

A solution mixture of 1 (0.30 g, 0.8 mmol),9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole-3-carbaldehyde (0.33 g, 1.1mmol) and piperidine (0.1 mL) in ethanol (40 mL) was heated to refluxovernight. After cooling down to room temperature, the organic solventwas removed. The residue was purified by precipitation from methanol andethyl acetate to afford SLG (0.25 g) in 48% yield. ¹H NMR (400 MHz,DMSO-d₆) δ 9.18 (d, J=6.8 Hz, 1H), 9.14 (d, J=8.0 Hz, 1H), 8.87 (s, 1H),8.55 (d, J=8.8 Hz, 1H), 8.51 (d, J=6.8 Hz, 1H), 8.45 (d, J=16 Hz, 1H),8.36 (d, J=16 Hz, 1H), 8.24 (d, J=7.2 Hz, 2H), 8.13 (d, J=7.6 Hz, 1H),8.05 (t, J=7.6 Hz, 1H), 7.77 (d, J=8.4 Hz, 1H), 7.69 (d, J=8.0 Hz, 1H),7.52 (t, J=7.2 Hz, 1H), 7.31 (t, J=7.2 Hz, 1H), 5.15 (t, J=5.2 Hz, 2H),4.63 (t, J=4.8 Hz, 2H), 3.97 (t, J=5.2 Hz, 2H), 3.84 (t, J=4.8 Hz, 2H),3.53 (t, J=4.8 Hz, 2H), 3.48 (t, J=4.8 Hz, 2H), 3.32-3.29 (m, 4H), 3.11(s, 3H), 3.06 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 153.5, 147.7, 145.3,142.2, 140.9, 138.1, 134.8, 128.8, 127.4, 126.7, 126.4, 122.8, 122.2,121.8, 120.4, 119.8, 119.2, 116.2, 114.8, 110.4, 110.3, 71.2, 71.1,69.8, 69.6, 68.8, 67.8, 58.1, 58.0, 55.9, 42.9. HRMS (MALDI-TOF) m/zCalcd for C₃₃H₃₇N₂O₄ 525.2747 Found 525.2747 [M]⁺.

3-Fluoro-9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole (5)

To a solution of 3-bromo-9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole (3.23g, 9.3 mmol) in dry THF (50 ml) was added n-BuLi (1.6 M, 8.7 ml, 13.9mmol) at −78° C. The resulting mixture was stirred for 50 min at −78° C.and then added with N-fluorobenzenesulfonimide (5.6 g, 18.6 mmol). Thereaction mixture was allowed to warm to rt and stirred for 2 h beforequenched with ammonia chloride solution. The organic layer wasseparated, dried over anhydrous sodium sulfate and evaporated undervacuum. The residue was purified by silica gel column chromatographyeluting with 3:1 petroleum ether/ethyl acetate to give compound 5 in 75%yield. ¹H NMR (400 MHz, CDCl₃) δ 8.03 (d, J=7.6 Hz, 1H), 7.73 (dd, J=2.4Hz, J=8.8 Hz 1H), 7.50-7.44 (m, 2H), 7.39 (dd, J=4.4 Hz, J=8.8 Hz, 1H),7.25-7.17 (m, 2H), 4.49 (t, J=6.4 Hz, 2H), 3.86 (t, J=6.4 Hz, 2H),3.52-3.50 (m, 2H), 3.43-3.41 (m, 2H), 3.32 (s, 3H). ¹³C NMR (100 MHz,CDCl₃) δ 158.6, 156.2, 141.4, 137.1, 126.3, 123.4, 123.3, 122.6, 122.6,120.6, 119.1, 113.6, 113.3, 109.7, 109.6, 109.2, 106.1, 105.9, 72.1,71.0, 69.4, 59.2, 43.4. HRMS (MALDI-TOF) m/z Calcd for C₁₇H₁₈FNO₂287.1316 Found 287.1314[M]⁺.

3-Bromo-6-fluoro-9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole (6)

To a solution of 3-fluoro-9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole(1.06 g, 3.71 mmol) in chloroform (20 ml) was added NBS (0.66 g, 3.71mmol) batch-wise in an ice-water bath. After complete addition, thereaction mixture was allowed to warm to room temperature slowly andstirred overnight. The reaction mixture was washed with water and brine.The organic layer was dried over anhydrous sodium sulfate and evaporatedunder reduced pressure to give compound 6 in 84% yield. ¹H NMR (400 MHz,CDCl₃) δ 8.09-8.06 (m, 1H), 7.63-7.60 (m, 1H), 7.52-7.50 (m, 1H),7.36-7.26 (m, 2H), 7.21-7.16 (m, 1H), 4.40 (d, J=5.6 Hz, 2H), 3.82-3.80(m, 2H), 3.49-3.46 (m, 2H), 3.40-3.38 (m, 2H), 3.29 (s, 3H). ¹³C NMR(100 MHz, CDCl₃) δ 158.7, 156.3, 140.1, 137.4, 128.9, 124.2, 124.2,123.2, 122.3, 122.2, 114.4, 114.1, 111.8, 110.8, 110.0, 109.9, 106.2,106.0. HRMS (MALDI-TOF) m/z Calcd for C₁₇H₁₇BrFNO₂ 366.0499 Found366.0502[M]⁺.

6-Bromo-9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole-3-carbaldehyde (7)

To a solution of3-bromo-6-fluoro-9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole (3.4 g, 9.3mmol) in dry THF (50 ml) was added n-BuLi (1.6 M, 8.7 ml, 13.9 mmol) at−78° C. The resulting mixture was stirred for 50 min at −78° C. and thenadded with N-formylmorpholine (1.86 ml, 18.6 mmol). The reaction mixturewas allowed to warm to rt and stirred for 2 h before quenched withammonia chloride solution. The organic layer was separated, dried overanhydrous sodium sulfate and evaporated under vacuum. The residue waspurified by silica gel column chromatography eluting with 2:1 petroleumether/ethyl acetate to give compound 7 in 65% yield. ¹H NMR (400 MHz,CDCl₃) δ 10.08 (s, 1H), 8.53 (s, 1H), 8.02-8.00 (m, 1H), 7.80-7.77 (m,1H), 7.54 (d, J=8.8 Hz, 1H), 7.45 (dd, J=4.0 Hz, J=9.2 Hz 1H), 7.28-7.23(m, 1H), 4.52 (t, J=5.6 Hz, 2H), 3.88 (t, J=5.6 Hz, 2H), 3.52-3.50 (m,2H), 3.40 (d, J=2.8 Hz, 2H), 3.28 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ191.8, 159.3, 156.9, 145.2, 137.8, 128.8, 127.5, 124.4, 123.7, 123.7,123.7, 122.8, 114.8, 114.5, 110.6, 110.5, 109.8, 106.7, 106.4, 72.1,71.0, 69.5, 59.2, 43.9. HRMS (MALDI-TOF) m/z Calcd for C₁₈H₁₈FNO₃316.1343 Found 316.1340[M]+.

(E)-4-(2-(6-Fluoro-9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)-1-(2-hydroxyethyl)quinolin-1-iumchloride (F-SLOH)

A solution mixture of 2 (0.21 g, 1.2 mmol),6-bromo-9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole-3-carbaldehyde (0.50g, 1.6 mmol) and piperidine (0.1 ml) in methanol (40 ml) was heated toreflux overnight. After being cooled down to room temperature, theorganic solvent was removed. The residue was purified by precipitationfrom methanol and ethyl acetate to afford F-SLOH in 65% yield. ¹H NMR(400 MHz, DMSO-d₆) δ 9.22 (d, J=6.8 Hz, 1H), 9.15 (d, J=8.4 Hz, 1H),8.91 (s, 1H), 8.58 (d, J=9.2 Hz, 1H), 8.53 (d, J=6.8 Hz, 1H), 8.40 (d,J=3.6 Hz, 1H), 8.24 (t, J=7.6 Hz 1H), 8.15-8.13 (m, 1H), 8.08-8.03 (m,2H), 7.78 (d, J=8.8 Hz, 1H), 7.74-7.71 (m, 1H), 7.40-7.35 (m, 1H), 5.34(s, 1H), 5.08-5.05 (m, 2H), 4.65-4.62 (m, 2H), 3.94-3.92 (m, 2H),3.84-3.91 (m, 2H), 3.46-3.45 (m, 2H), 3.31-3.29 (m, 2H), 3.10 (s, 3H).¹³C NMR (100 MHz, DMSO-d₆) δ 168.6, 154.3, 145.2, 143.4, 142.1, 142.0,140.9, 126.5, 126.3, 122.9, 122.8, 122.7, 122.1, 121.4, 120.4, 119.9,110.6, 110.3, 71.3, 69.9, 68.9, 58.1. HRMS (MALDI-TOF) m/z Calcd forC₃₀H₃₀FN₂O₃ 485.2235 Found 485.2211 [M]⁺.

(E)-1-(2-Ethoxy-2-oxoethyl)-4-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)quinolin-1-iumbromide (SLAce)

A solution of 8 (0.21 g, 0.5 mmol) and ethyl 2-bromoacetate (0.33 g, 2.0mmol) in ethanol was stirred overnight at room temperature. Aftersolvent removal, the residue was precipitated from methanol and ethylacetate to afford SLAce (0.15 g) in 52% yield. ¹H NMR (400 MHz, DMSO-d₆)δ 9.24 (d, J=6.8 Hz, 1H), 9.18 (d, J=8.4 Hz, 1H), 8.89 (s, 1H), 8.61 (d,J=6.8 Hz, 1H), 8.53 (d, J=16 Hz, 1H), 8.42 (d, J=16 Hz, 1H), 8.31 (d,J=8.8 Hz, 1H), 8.26-8.23 (m, 2H), 8.15 (d, J=8.4 Hz, 1H), 8.06 (t, J=7.6Hz, 1H), 7.80 (d, J=8.4 Hz, 1H), 7.72 (d, J=8.0 Hz, 1H), 7.53 (t, J=8.0Hz, 1H), 7.33 (t, J=7.2 Hz, 1H), 5.99 (s, 2H), 4.64 (t, J=5.2 Hz, 2H),4.25 (tr, J=7.2 Hz, 2H), 3.84 (t, J=5.2 Hz, 2H), 3.47 (m, 2H), 3.31 (m,2H), 3.11 (s, 3H), 1.26 (t, J=5.2 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ166.5, 154.6, 147.9, 146.5, 142.4, 140.9, 138.7, 128.9, 127.6, 126.7,126.6, 126.0, 122.8, 122.2, 122.1, 120.4, 119.9, 118.9, 116.2, 115.0,110.5, 110.4, 71.3, 69.8, 68.8, 62.3, 58.1, 56.4, 42.9, 13.9. HRMS(MALDI-TOF) m/z Calcd for C₃₂H₃₃N₂O₄ 509.2446 Found 509.2427 [M]⁺.

(E)-1-(2-Amino-2-oxoethyl)-4-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)quinolin-1-iumbromide (SLAD)

A solution of 8 (0.21 g, 0.5 mmol) and 2-bromoacetamide (0.27 g, 2.0mmol) in acetonitrile was heated to reflux overnight. After removing thesolvent, the residue was precipitated from methanol and ethyl acetate toafford SLAD (0.15 g) in 63% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 9.24 (d,J=6.8 Hz, 1H), 9.17 (d, J=8.4 Hz, 1H), 8.89 (s, 1H), 8.58 (d, J=6.8 Hz,1H), 8.45 (dd, J=33.6 Hz, J=18 Hz, 2H), 8.28-8.25 (m, 2H), 8.18-8.14 (m,3H), 8.08-8.04 (m, 1H), 7.80 (d, J=8.4 Hz, 2H), 7.72 (d, J=8.4 Hz, 1H),7.55-7.51 (m, 1H), 7.34-7.31 (m, 1H), 5.68 (s, 2H), 4.66-4.63 (m, 2H),3.86-3.83 (m, 2H), 3.49-3.47 (m, 2H), 3.31-3.29 (m, 2H), 3.11 (s, 3H).¹³C NMR (100 MHz, DMSO-d₆) ⁸ 166.3, 154.0, 148.2, 145.8, 142.3, 140.9,138.7, 135.2, 128.8, 127.5, 126.7, 126.4, 126.1, 122.8, 122.2, 122.0,120.4, 118.5, 116.2, 115.0, 110.5, 110.4, 71.3, 69.8, 68.8, 58.1, 57.8,42.9. HRMS (MALDI-TOF) m/z Calcd for C₃₀H₃₀N₃O₃ 480.2281 Found 480.2301[M]⁺.

In summary, carbazole-based fluorophores bind to Aβ(1-40) and Aβ(1-42)as well as Aβ aggregates with strong fluorescence enhancement, thusallowing their direct imaging and labeling. TIRFM technique is used tostudy the effects of these molecules on Aβ aggregation/fibrillation.Some carbazole-based fluorophores, for instance, F-SLOH, and SLAD arenon-toxic, potent Aβ aggregation inhibitors and exhibit a protectiveeffect against the neurotoxic activities of the Aβ oligomers and fibrilstowards neuronal cells. These properties of F-SLOH, and SLAD togetherwith their ability to cross the blood-brain barrier and target the Aβplaques, render their application for neuroprotective therapy and astherapeutic agent for Alzheimer's disease.

If desired, the different functions discussed herein may be performed ina different order and/or concurrently with each other. Furthermore, ifdesired, one or more of the above-described functions may be optional ormay be combined.

While the foregoing invention has been described with respect to variousembodiments and examples, it is understood that other embodiments arewithin the scope of the present invention as expressed in the followingclaims and their equivalents. Moreover, the above specific examples areto be construed as merely illustrative, and not limitative of thereminder of the disclosure in any way whatsoever. Without furtherelaboration, it is believed that one skilled in the art can, based onthe description herein, utilize the present invention to its fullestextent. All publications recited herein are hereby incorporated byreference in their entirety.

INDUSTRIAL APPLICABILITY

The objective of the presently claimed invention is to provide methodsfor labeling, imaging and detecting the beta-amyloid (Aβ) peptides,oligomers, and fibrils in vitro by using carbazole-based fluorophores. Afurther aspect of the present invention relates to a method of reducingand preventing aggregation of beta-amyloid peptides for Alzheimer'sdisease (AD) as well as of treating and/or preventing Alzheimer'sdisease by using carbazole-based fluorophores.

What is claimed is:
 1. A method of labeling, imaging and detectingbeta-amyloid (Aβ) peptides, oligomers and fibrils using carbazole-basedfluorophores comprising a formula S or V series:

wherein Ar is a heteraromatic ring selected from the group consisting ofpyridinyl, substituted pyridinyl, quinolinyl, substituted quinolinyl,acridinyl, substituted acridinyl, benzothiazolyl, substitutedbenzothiazolyl, benzoxazolyl, and substituted benzoxazolyl; R₁ isselected from the group consisting of polyethylene glycol chain, alkyl,substituted alkyl, peptide chain, glycosidyl, andC(O)NHCH((CH₂CH₂O)₂CH₃)₂; R₂ is selected from the group consisting ofethenyl, ethynyl, azo and azomethinyl. R₃ is selected from the groupconsisting of alkyl, HO-alkyl, HS-alkyl, H₂N-alkyl, HN-alkyl-alkyl,HOOC-alkyl, (alkyl)₃N⁺-alkyl, and (Ph)₃P⁺-alkyl; and X is an anionselected from the group consisting of F⁻, Cl⁻, Br⁻, I⁻, HSO₄ ⁻, H₂PO₄ ⁻,HCO₃ ⁻, tosylate, and mesylate.
 2. The method according to claim 1,wherein said Ar is selected from a quinolinyl or substituted quinolinyl;said R₁ is a 2-(2-methoxyethoxy)ethoxy; said R₂ is an ethenyl; said R₃is selected from a methyl, 2-hydroxyethyl, ethyl or 3-hydroxypropyl; andsaid X is selected from a chloride, bromide or iodide, thecarbazole-based fluorophores thereof are represented by the formula SLM,SLOH, SLE and SLOH-Pr:


3. The method according to claim 1, wherein said Ar is selected from aquinolinyl or substituted quinolinyl; said R₁ is a methyl; said R₂ is anethenyl; said R₃ is a methyl; and said X is selected from a chloride,bromide or iodide, and the carbazole-based fluorophores thereof arerepresented by the formula Me-SLM:


4. The method according to claim 1, wherein said Ar is selected from anacridinyl or substituted acridinyl; said R₁ is a2-(2-methoxy-ethoxy)ethoxy; said R₂ is an ethenyl; said R₃ is selectedfrom a methyl or 2-hydroxyethyl; and said X is selected from a chloride,bromide or iodide, and the carbazole-based fluorophores thereof arerepresented by the formula SAM and SAOH:


5. The method according to claim 1, wherein said Ar is selected from apyridinyl or substituted pyridinyl; said R₁ is a2-(2-methoxyethoxy)ethoxy; said R₂ is an ethenyl; said R₃ is selectedfrom a methyl or 2-hydroxyethyl; and said X is selected from a chloride,bromide or iodide, the carbazole-based fluorophores thereof arerepresented by the formula SPM and SPOH:


6. The method according to claim 1, wherein said carbazole-basedfluorophores are conjugated with paramagnetic metal complexes comprisinggadolinium(III), iron(III), manganese(II) complexes, Gd(III)-basedchelates, and any complexes which are detected by magnetic resonanceimaging.
 7. The method according to claim 1, wherein saidcarbazole-based fluorophores are used in imaging techniques comprisingmagnetic resonance imaging, positron emission tomography, near-infraredfluorescence imaging and multiphoton excited imaging.
 8. The methodaccording to claim 1, wherein the carbazole-based fluorophores arewater-soluble.
 9. The method according to claim 1, wherein thecarbazole-based fluorophores are non-toxic.
 10. The method according toclaim 1, wherein the carbazole-based fluorophores are able to passthrough the blood-brain barrier.
 11. The method according to claim 1,further comprising introducing the carbazole-based fluorophores tosample being labeled, imaged and detected in vitro.
 12. The methodaccording to claim 1, further comprising administering thecarbazole-based fluorophores in vivo.
 13. A method of labelling, imagingand detecting beta-amyloid (Aβ) peptides, oligomers, and fibrils usingcarbazole-based fluorophores comprising a formula S series:

wherein Ar is a heteraromatic ring selected from the group consisting ofpyridinyl, substituted pyridinyl, quinolinyl, substituted quinolinyl,acridinyl, substituted acridinyl, benzothiazolyl, substitutedbenzothiazolyl, benzoxazolyl, and substituted benzoxazolyl; R₁ is aradical selected from the group consisting of polyethylene glycol chain,alkyl, substituted alkyl, peptide chain, glycosidyl, andC(O)NHCH((CH2CH2O)2CH3)2; R₂ is selected from the group consisting ofethenyl, ethynyl, azo and azomethinyl. R₃ is a radical selected from thegroup consisting of HO-alkyl, alkyl-COOalkyl, alkyl-CONH2,Alkyl-CONHalkyl, Polyethylene Glycol Chain; X is an anion selected fromthe group consisting of F, Cl, Br, I, HSO4, H2PO4, HCO3, tosylate, andmesylate; Y is selected from the group consisting of H, F, Cl, OH, andOCH3.
 14. The method according to claim 13, wherein Ar is selected froma quinolinyl or substituted quinolinyl; said R1 is a2-(2-methoxyethoxy)ethoxy; said R2 is an ethenyl; R3 is a 2-hydroxyethylor acetamide or acetate or 2-(2-methoxyethoxy)ethoxy; said X is achloride or bromide or iodide and said Y is a H or F which arerepresented by the formula F-SLOH, SLAD, SLAce, and SLG:


15. The method according to claim 13, wherein said carbazole-basedflorophores are conjugated with paramagnetic metal complexes comprisinggadolinium(III), iron(III), manganese(II) complexes, Gd(III)-basedchelates, and any complexes which are detected by magnetic resonanceimaging.
 16. The method according to claim 13, wherein saidcarbazole-based fluorophores are used in imaging techniques comprisingmagnetic resonance imaging, positron emission tomography, near-infraredfluorescence imaging and multiphoton excited imaging.
 17. The methodaccording to claim 13, wherein the carbazole-based fluorophores arenon-toxic.
 18. The method according to claim 13, wherein thecarbazole-based fluorophores are able to pass through the blood-brainbarrier.
 19. The method according to claim 13, further comprisingintroducing the carbazole-based fluorophores to sample being labeled,imaged and detected in vitro.
 20. The method according to claim 13,further comprising administering the carbazole-based fluorophores invivo.